Abstract
We describe Ribo Mega-SEC, a powerful approach for the separation and biochemical analysis of mammalian polysomes and ribosomal subunits using Size Exclusion Chromatography and uHPLC. Using extracts from either cells, or tissues, polysomes can be separated within 15 min from sample injection to fraction collection. Ribo Mega-SEC shows translating ribosomes exist predominantly in polysome complexes in human cell lines and mouse liver tissue. Changes in polysomes are easily quantified between treatments, such as the cellular response to amino acid starvation. Ribo Mega-SEC is shown to provide an efficient, convenient and highly reproducible method for studying functional translation complexes. We show that Ribo Mega-SEC is readily combined with high-throughput MS-based proteomics to characterize proteins associated with polysomes and ribosomal subunits. It also facilitates isolation of complexes for electron microscopy and structural studies.
| Original language | English |
|---|---|
| Article number | e36530 |
| Pages (from-to) | 1-26 |
| Number of pages | 26 |
| Journal | eLife |
| Volume | 7 |
| Issue number | 7 |
| DOIs | |
| Publication status | Published - 10 Aug 2018 |
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Keywords
- Amino Acids/chemistry
- Animals
- Cell Line
- Chromatography, High Pressure Liquid
- Humans
- Mice
- Polyribosomes/chemistry
- Protein Biosynthesis
- Proteomics
- Ribosomes/chemistry
Cite this
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Efficient Analysis of Mammalian Polysomes in cells and tissues using Ribo Mega-SEC. / Yoshikawa, Harunori; Larance, Mark; Harney, Dylan J.; Sundaramoorthy, Ramasubramanian; Ly, Tony; Owen-Hughes, Thomas; Lamond, Angus I. (Lead / Corresponding author).
In: eLife, Vol. 7, No. 7, e36530, 10.08.2018, p. 1-26.Research output: Contribution to journal › Article
TY - JOUR
T1 - Efficient Analysis of Mammalian Polysomes in cells and tissues using Ribo Mega-SEC
AU - Yoshikawa, Harunori
AU - Larance, Mark
AU - Harney, Dylan J.
AU - Sundaramoorthy, Ramasubramanian
AU - Ly, Tony
AU - Owen-Hughes, Thomas
AU - Lamond, Angus I.
N1 - Wellcome; H2020 | H2020 Priority Excellent Science | H2020 Marie Skłodowska-Curie Actions (MSCA); Uehara Memorial Foundation; Naito Foundation; Royal Society of Edinburgh (RSE)
PY - 2018/8/10
Y1 - 2018/8/10
N2 - We describe Ribo Mega-SEC, a powerful approach for the separation and biochemical analysis of mammalian polysomes and ribosomal subunits using Size Exclusion Chromatography and uHPLC. Using extracts from either cells, or tissues, polysomes can be separated within 15 min from sample injection to fraction collection. Ribo Mega-SEC shows translating ribosomes exist predominantly in polysome complexes in human cell lines and mouse liver tissue. Changes in polysomes are easily quantified between treatments, such as the cellular response to amino acid starvation. Ribo Mega-SEC is shown to provide an efficient, convenient and highly reproducible method for studying functional translation complexes. We show that Ribo Mega-SEC is readily combined with high-throughput MS-based proteomics to characterize proteins associated with polysomes and ribosomal subunits. It also facilitates isolation of complexes for electron microscopy and structural studies.
AB - We describe Ribo Mega-SEC, a powerful approach for the separation and biochemical analysis of mammalian polysomes and ribosomal subunits using Size Exclusion Chromatography and uHPLC. Using extracts from either cells, or tissues, polysomes can be separated within 15 min from sample injection to fraction collection. Ribo Mega-SEC shows translating ribosomes exist predominantly in polysome complexes in human cell lines and mouse liver tissue. Changes in polysomes are easily quantified between treatments, such as the cellular response to amino acid starvation. Ribo Mega-SEC is shown to provide an efficient, convenient and highly reproducible method for studying functional translation complexes. We show that Ribo Mega-SEC is readily combined with high-throughput MS-based proteomics to characterize proteins associated with polysomes and ribosomal subunits. It also facilitates isolation of complexes for electron microscopy and structural studies.
KW - Amino Acids/chemistry
KW - Animals
KW - Cell Line
KW - Chromatography, High Pressure Liquid
KW - Humans
KW - Mice
KW - Polyribosomes/chemistry
KW - Protein Biosynthesis
KW - Proteomics
KW - Ribosomes/chemistry
U2 - 10.7554/eLife.36530
DO - 10.7554/eLife.36530
M3 - Article
VL - 7
SP - 1
EP - 26
JO - eLife
JF - eLife
SN - 2050-084X
IS - 7
M1 - e36530
ER -